Inflammatory bowel disease (IBD) affects as many as 100 per 100,000 Americans under the age of 30. Recent studies suggest that IBD may be caused by alterations in the delicate balance between pro- and anti- inflammatory cytokines in the gastro-intestinal (GI) tract. The role of tumor necrosis factor-alpha (TNF) and interleukin-10 (IL-10) as pro- and anti-inflammatory cytokines, respectively, has been examined in preclinical and clinical studies. In the proposed study, an interdisciplinary team of investigators will evaluate oral gene therapy using soluble TNF receptor-1 (sTNF-R1) and IL-10 expressing plasmid DNA in acute colitis-induced Balb/c mice. The plasmid DNA will be encapsulated in gelatin nanoparticles, which are further protected by a poly(epsilon- caprolactone (PCL) matrix to form Nanoparticles-in-Microsphere Oral System (NiMOS) of 1-5 mm in diameter. Our innovative strategy using non-viral gene delivery vector relies on the fact that gelatin nanoparticles can efficiently encapsulate plasmid DNA, are internalized in cells by endocytosis, protects against DNA degradation in cells, and allows for efficient transfection. The PCL matrix is expected to protect the DNA- containing nanoparticles upon oral administration and allow the release to occur in the small and large intestine due to enzymatic degradation with lipases. Our preliminary studies show that optimized NiMOS formulation have longer residence time in the small and large intestine upon administration in fasted rats and can afford transfection of reporter plasmids in the small and large intestine. The specific aims of this R01 application are: (1) to prepare, characterize, and optimize NiMOS formulations containing plasmid DNA encoding for sTNF-R1 and IL-10, (2) evaluate the biodistribution pattern and residence profile of NiMOS upon oral administration in naove and acute colitis-induced Balb/c mice, (3) determine qualitative and quantitative transfection efficiency upon and oral and rectal administration of DNA- containing NiMOS in naove and colitis-induced mice, and (4) evaluate the therapeutic efficacy of expressed sTNF-R1 and IL-10 in acute colitis-induced mice upon oral and rectal administration. This study will provide important understanding of the barriers to oral therapeutic gene delivery and the potential of NiMOS as safe and effective non-viral vector system. The potential of oral gene therapy for the treatment of IBD using this clinically-translatable strategy has tremendous promise in the effective management of this disease. Project Narrative: Inflammatory bowel disease (IBD) is a significant clinical problem affecting as many as 100 per 100,000 Americans under the age of 30. Recent studies suggest that IBD may be caused by the alternations in the delicate balance between pro- and anti-inflammatory protein molecules, called cytokines, in the gastro- intestinal (GI) tract. Due to the presence of proteolytic degrading enzymes, oral administration of protein drugs does not provide an effective therapy. In the proposed study, an interdisciplinary group of investigators will evaluate if oral gene therapy with plasmid DNA that encodes for soluble receptor (soluble tumor necrosis factor receptor-1, sTNF-R1) and anti-inflammatory cytokine interleukin-10 (IL-10) can be used as a clinically translatable alternative for treatment of IBD. We have formulated Nanoparticles-in-Microsphere Oral System (NiMOS) to provide a safe and effective gene delivery mechanism to the small and large intestine upon oral administration. The sTNF- R1- and IL-10-encoding plasmid DNA will be encapsulated in gelatin nanoparticles, which are further protected with a poly(epsilon-caprolactone) (PCL) matrix to form 1-5 micron particles for effective delivery. Once the NiMOS reach small and large intestine, the PCL shell is degraded and the DNA-containing gelatin nanoparticles are released for cellular uptake and efficient gene expression. Our preliminary studies show that NiMOS, optimized for oral gene delivery, can localize the encapsulated nanoparticles in the small and large intestine in fasted rats and lead to transfection of the encapsulated reporter plasmid. In this study we will develop NiMOS with plasmid DNA encoding for sTNF-R1 and IL-10, characterize, and optimize them for oral delivery. Following the optimization of DNA-containing NiMOS, we will examine distribution and residence of NiMOS upon oral administration in naove and colitis-induced Balb/c mice. The qualitative and quantitative transfection efficiency of sTNF-R1 and IL-10 will be examined next. Lastly, we will carefully evaluate the therapeutic effectiveness of orally-administered NiMOS containing plasmid DNA that encodes for sTNF-R1 and IL-10 in colitis-induced mice. The results of this study will be extremely beneficial, since the expressed therapeutic protein can be produced locally at the disease site for a long period of time. In addition, NiMOS would be useful for other oral gene therapy strategies for treatment of diseases or oral DNA vaccination.